Cardiovascular disease is one of the major life-threatening diseases in Western society, but biomarkers to monitor the severity or the progression of the disease are presently not available. Also, the number of biochemically detectable risk factors (e.g., serum cholesterol, triglycerides, ApoE genotype) is surprisingly low.
Vitamin K is a cofactor in the posttranslational conversion of glutamate residues into γ-carboxyglutamate (Gla). At this time, 10 mammalian Gla-containing proteins have been described in detail, and the number of Gla-residues per molecule varies from 3 (osteocalcin) to 13 (protein Z). In all cases in which their function was known, the activity of the various Gla-proteins was strictly dependent on the presence of the Gla-residues (Shearer, M. J., Brit. J. Haematol. (1990) 75:156-162; Vermeer, C., Biochem. J. (1990) 266:625-636). Gla-proteins are synthesized in various tissues, for instance the liver, bone and vessel wall. Blood coagulation factors 11 (prothrombin), VII, IX and X are examples of Gla-proteins synthesized in the liver, examples of so-called extrahepatic Gla-proteins are osteocalcin and Matrix Gla-Protein (Hauschka, P. V. et al., Phys. Rev. (1989) 69:990-1047).
Matrix Gla-Protein is a vitamin K-dependent protein synthesized in bone and in a number of soft tissues including heart and vessel wall. In experimental animals its soft tissue expression is high immediately after birth, but decreases in the months thereafter. Only in cartilage and arteries does its expression seem to continue lifelong. Although the precise function of MGP on a molecular level has remained unknown so far, experiments with MGP-deficient transgenic animals (“knock-out” mice) have shown that MGP has a prominent role in the prevention of vascular mineralization: MGP-deficient animals are born to term but develop severe aortic calcification (as analyzed by X-ray) in the first weeks of life; and eventually all animals die within 6-8 weeks after birth due to rupture of the aorta or one of the other main arteries (Luo, G. et al., Nature (1997) 386:78-81).
MGP contains five Gla-residues which are essential for its calcification-inhibitory function. This was discovered by Price et al., who treated rats with the vitamin K-antagonist warfarin thus blocking the formation of Gla-residues (Price, P. A. et al. Arterioscler. Thromb. Vasc. Biol. (1998) 18: 1400-1407). The treatment protocol induced vitamin K-deficiency in the vasculature and lead to vascular calcification within 3 weeks. Hence an inadequate vitamin K-status leading to incomplete MGP carboxylation is a risk factor for cardiovascular calcification. Conformation-specific antibodies recognizing either carboxylated (=active conformation containing 5 Gla-residues/mol) or under-carboxylated (=inactive conformation containing less than 5 Gla-residues/mol) are a powerful tool in the diagnostics of cardiovascular disease.
MGP was discovered in bone (Price, P. A. et al., Biochem. Biophys. Res. Commun. (1983) 117:765-771), but in situ hybridization experiments showed that it is also expressed in other tissues including the vessel wall (Fraser, D. J. et al., J. Biol. Chem. (1988) 263:11033-11036). With polyclonal antibodies raised against a synthetic peptide homologous to the C-terminus of bovine MGP, the protein was also found in cartilage via immunohistochemical staining (Loeser, R. F. et al., Biochem. J. (1992) 282:1-6). A radioimmunoassay was developed for the detection of serum MGP in the rat, but in these experiments circulating MGP was correlated with maturation of rat bone, and not with vascular biology (Otawara, Y. et al., J. Biol. Chem. (1986) 261:10828-10832).
Research concerning the role of MGP in the vessel wall has not started before the discovery by Luo et al. (supra) that MGP is a strong inhibitor of vascular calcification in mice. Since then, evidence has accumulated suggesting that bone calcification and atherosclerotic vessel wall calcification proceed via very similar mechanisms, in which the same proteins (including MGP) are used (Proudfoot, D. et al., Arterioscler. Thromb. Vasc. Biol. (1998) 18:379-388; Proudfoot, D. etal., J. Pathol. (1998) 185:1-3). Most studies on the regulation of MGP expression have been performed in smooth muscle cell cultures, with mRNA detection as a measure for MGP synthesis. Recent studies in humans have shown that, although MGP mRNA is constitutively expressed by normal vascular smooth muscle cells, it is substantially upregulated in cells adjacent to both medial and intimal calcification (Shanahan, C. M. et al., Crit. Rev. in Eukar. Gene Expr. (1998) 8:357-375).
The prior art neither teaches nor suggests the use of MGP as a marker for angiogenesis or cardiovascular disease, or the like, nor does it disclose an assay for circulating MGP in humans.
As stated above, biomarkers to monitor the severity or the progression of cardiovascular disease are not available up till now, and the number of biochemically detectable risk factors is very low. Therefore, there is clearly a need for biomarkers for vascular characteristics, for assessment of the severity or progression of atherosclerosis and related diseases, as well as for monitoring the effect of treatment during vascular disease.